New directions in bacterial vaccine discovery may arise from studies of host-microbe interactions, particularly through the use of a newly described technology: the dynamic in vitro attachment and invasion system (DIVAS). DIVAS was developed to study bacterial attachment and invasion with cells held at specific and controlled conditions of growth, metabolism, and nutrient levels. Results from experiments performed with DIVAS and group B Streptococcus (GBS) type III strains substantiated earlier findings that capsular polysaccharide is not critical for invasion of respiratory epithelial cells. Moreover, GBS invaded these cells only when held at a fast as opposed to a relatively slower rate of growth and they expressed several proteins solely under growth conditions conducive for invasion. In this proposal, we seek to test the hypothesis that GBS proteins involved with invasion of eukaryotic cells are new and important targets of protective immunity. GBS is a major cause of neonatal sepsis and meningitis, and is increasingly prevalent among non-pregnant adults and the elderly with underlying illnesses. Preclinical and clinical trials have been successfully performed with protein conjugate vaccines prepared with GBS polysaccharides. GBS proteins with virulence properties have been described and some with vaccine potential have been tested preclinically. In this proposal, we will use DIVAS to identify physiological conditions conducive for bacterial attachment/invasion of eukaryotic cells using GBS as a model pathogen. We will isolate and identify GBS membrane proteins expressed solely under invasive conditions (Sp. Aim 1). GBS mutants lacking genes for these proteins will be constructed and tested for invasiveness in DIVAS and virulence in mice (Sp. Aim 1). Several of these proteins will be purified directly from GBS, or cloned and recombinantly expressed, and tested as vaccine candidates in mice (Sp. Aim 2). Findings from these studies utilizing this unique approach to vaccine antigen discovery could be directly applied to other bacterial pathogens.